Exposure to osmotic stress increases the insertion of AQP5 in the membranes of cortical fiber cells: implications for the water permeability of lens fiber cells

Abstract

Purpose: To develop methods to determine whether changes in the sub-cellular distribution of the water channel, AQP5, observed during lens differentiation, or in response to changes in extracellular osmolarity alter the water permeability (P_H2O) of lens fiber cells.<br />

Methods: Fresh lenses or lenses pre-incubated in Artificial Aqueous Humor of varying osmolarity (270-405mOsm) were either fixed for immunohistochemistry¹ or separated into epithelial cells and fiber cell vesicles preparations². Confocal imaging of immunolabelled lens cryosections and membrane vesicles was used to determine changes in the sub-cellular distribution of AQP5 under different incubation conditions in different lens regions. Isolated epithelial cells or fiber cell vesicles were loaded with the fluorescent dye Calcein AM and the rate of change in fluorescence intensity in response to an applied osmotic gradient, plus the surface area of the cells used to calculate P_H2O. Functional experiments were performed in the presence or absence of HgCl that preferentially blocks AQP1 and AQP5, but not AQP0.<br />

Results: A change in the sub-cellular distribution of AQP5 from the cytoplasm to the cell membrane was observed in the outer cortex of the rat lenses, but was restricted to only superficial layers of fiber cells in the mouse and bovine lenses. Within these zones of cytoplasmic AQP5 labelling the incubation of lenses in hypoosmotic solutions resulted in an increased insertion of AQP5 into fiber cell membranes. Functional experiments on acutely isolated rat lenses revealed that epithelial cells contain Hg⁺-sensitive water channels consistent with the expression of AQP1 and 5 in these cells. However, rat fiber cell vesicles were not sensitive to Hg⁺, a result consistent with the observed sub-cellular location of AQP5 in the cytoplasm.<br />

Conclusions: Our immunolocalization results suggest fiber cells can respond to osmotic stress by shuttling AQP5 to and from the cell membrane to dynamically regulate the overall P_H2O of the lens. To test this hypothesis we have developed and validated a fluorescence assay that can be used to monitor changes in P_H2O in fiber cell vesicles obtained from lenses incubated under different experimental conditions.<br /> Supported by NIH (Grant #EY13462).<br /> 1. Jacobs et al. Microsc Res Tech. 62:83-91, 2003.<br /> 2. Varadaraj et al. J Membr Biol. 170:191-203, 1999.<br />